Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.     

Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines

Three cell lines of Heliothis zea and one cell line of Heliothis virescens replicated the singleembedded, nuclear polyhedrosis virus (NPV) of H. Zea, (i.e., Baculovirus heliothis) with concomitant production of polyhedral inclusion bodies (PIB). Between 20 and 60% of the H. zea cells produced PIB, whereas only 3% of H. virescens cells were found to produce PIB. The H. zea cell lines produced 10 to 20 times more PIB than did the H. virescens cell line. The PIB from all cell lines produced typical symptoms of an NPV infection when bioassayed against larvae of H. zea. More than 99% of the total viral activity of the final whole culture was due to the PIB.     

Pharmacokinetics of chlorogenic acid and rutin in larvae of Heliothis zea

Studies using [³H]chlorogenic acid and [³H]rutin demonstrated that the kinetics of uptake of these plant phenolics into the haemolymph of 5th-instar Heliothis zea (Boddie) following actue oral administration is a first-order process. The total quantity of either phenolic present in the haemolymph within 1 hr amounts to 5% or less of the total ingested dose. Based on TLC analyses, 80% or more of the radioactivity in the haemolymph occurs as the parent phenolic. Retention of [³H]-chlorogenic acid or [³H]-rutin in H. zea following chronic feeding from 1st to 3rd-instar larvae is also linearly related to dietary dose. Chlorogenic acid and rutin are both equitoxic and equivalent in bioavailability to H. zea.Loss of [³H]-rutin from the haemolymph of 5th-instar larvae following injection is biphasic. One half of the injected dose is excreted in the frass in the first 6 hr after injection; the other half is thereafter eliminated at 1/20th of the initial rate. Analyses of extracts of frass by thin-layer chromatography indicate that after either chronic or acute feeding 90% of the ingested phenolic is excreted unchanged. Possible sites and modes of action of phenolics in insects are discussed in light of these findings.     

Selection for resistance to a nucleopolyhedrosis virus in laboratory populations of the cotton bollworm, Heliothis zea

Resistance to a nucleopolyhedrosis virus (Baculovirus heliothis) did not develop in laboratory populations of the cotton bollworm, Heliothis zea. A selection pressure of LD₅₀ to 70 was maintained throughout 20 to 25 generations of selection. No significant changes in LD₅₀, slope, or intercept of dose-mortality lines were detected. Laboratory populations under selection were as susceptible to the virus as nonselected or wild populations of H. zea. The resistance ratio (LD₅₀ of selected generation/initial generation) ranged from 0.5 to 1.2.     

Interactions of the microsporidium Vairimorpha necatrix with a bacterium,virus, and fungus in Heliothis zea

Interactions between Vairimorpha necatrix and three other pathogens of Heliothis zea were evaluated with dose-mortality studies in order to find a synergistic combination which could be tested for field control of H. zea. The effect of the microsporidium combined with the bacterium Bacillus thuringiensis was at least additive, with indications of synergism. The interaction between V. necatrix and Heliothis nuclear-polyhedrosis virus was antagonistic except that the highest microsporidian dose overcame the antagonism with a resultant independent action. The interaction between V. necatrix and the fungus Nomuraea rileyi was additive, though response varied some-what with different proportions of the two pathogens. Even though none of these interactions is likely to be valuable in microbial control, V. necatrix has the potential to synergize or antagonize any biological or chemical agent that acts on the midgut epithelium of host insects.     

Quantification of the dose-Mortality response of Trichoplusia ni,Helioithis, zea,and Spodoptera frugiperda to Nuclear polyhedrosis viruses: Applicability of an exponential model

In vivo assays of several nuclear polyhedrosis viruses were conducted in neonate Trichoplusia ni, Heliothis zea, and Spodoptera frugiperda larvae using a droplet feeding method. Characteristic deviations from the probit model were observed with several virus isolats that suggested the dose-reponse relation might be determined primarily by the chance of obtaining virus from the inoculum rather than by variabilityin host susceptibility. This was supported by comparisons of the slopes of the probit lines with the corresponding expected values based on the exponential relationship described by a one-hit Poissonian curve. Where applicable, the exponential model offers severl advantages over the probit model in describing and quantifying virus-host relations.     

Temperature-sensitive mechanism regulating diapause in Heliothis zea

Pupal diapause in Heliothis zea is regulated by a temperature-sensitive mechanism which prevents ecdysone production despite the release of prothoracicotropic hormone. To determine how this mechanism functioned, donor prothoracic glands were implanted into prothoracic gland-ablated hosts to test their ability to produce ecdysone in a diapause-sustaining temperature of 19°C. Results of these experiments ruled out the possibility that ecdysis production was regulated by the nervous system or by a mechanism intrinsic to the prothoracic glands, and suggested that a humoral factor was required for diapause termination.Haemolymph injection experiments supported this humoral factor hypothesis, i.e. haemolymph from non-diapausing donor pupae terminated diapause in hosts maintained at 19°C, whereas haemolymph from diapausing donor pupae had no such effect. These findings indicate that the temperature-sensitive mechanism regulating H. zea diapause functions by controlling the availability of a humoral factor necessary for ecdysone production by the prothoracic glands.     

Characterization of the polyhedral envelope of the nuclear polyhedrosis virus of Heliothis virescens

The polyhedral envelope of the nuclear polyhedrosis virus of Heliothis virescens was separated from the matrix proteins and nucleocapsids by alkaline dissolution and differential centrifugation. Spectrophotometric analyses and histochemical staining demonstrated that the envelope was composed of carbohydrates. The envelope contained 60.9% (by weight) hexose and 29.1% pentose. Further examination revealed significant amounts of uronic acids (8.4%) and hexosamines (1.6%).     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

Survival of the nuclear polyhedrosis virus of Heliothis armigera on crops and in soil in Botswana

Experiments are described that record the change in activity of the nuclear polyhedrosis virus of Heliothis armigera on cotton and sorghum in the short-term and long-term survival of the virus on sorghum and in soil. Virus activity was lost rapidly on cotton but remained at a high level for up to 30 days on sorghum. Activity was still detectable on harvested sorghum more than 80 days after spraying and on the soil below, but soil activity fell to less than one-third of its original level during the winter.     

Effects of cytoplasmic polyhedrosis virus on diapausing Heliothis virescens

Laboratory studies were conducted to determine effects of cytoplasmic polyhedrosis virus on diapausing Heliothis virescens. Most virus-infected individuals died in the larval stage. Infected pupae yielded as many moths as healthy. Females from surviving virus-infected larvae produced fewer eggs than those from healthy larvae, but there was no statistical difference in longevity of adults between healthy and infected groups. Infected moths yielded lower than normal quantities of extracted fatty acids.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Comparative studies on the nuclear polyhedrosis viruses of Lymantria monacha and L. dispar

Immunodiffusion and tube precipitation tests, polyacrylamide gel electrophoresis of virus polypeptides, and cross-transmission experiments suggest that two nuclear polyhedrosis viruses, one from Lymantria monacha and one from L. dispar, are partially related to each other, but not identical. The virus particle proteins seem to be more specific than the polyhedron proteins.     

Changes in growth and virulence of a nuclear polyhedrosis virus from Choristoneura fumiferana after passage in Trichoplusia ni and Galleria mellonella

An isolate of nuclear polyhedrosis virus from Choristoneura fumiferana was fed to neonate larvae of Trichopulsia ni and Galleria mellonella. It caused infection and mortality in both of these species. After passage in the alternate hosts, the isolate became increasingly virulent for these hosts. The passaged virus retained its infectivity for Choristoneura but diseased larvae did not wilt and at death they were found to contain only a few polyhedra indicating the virus had been changed.     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.     

Comparative peptide mapping of baculovirus polyhedrins

Amino terminals and two-dimensional high-voltage peptide maps of tryptic digests of polyhedrins from Heliothis armigera nuclear polyhedrosis virus (NPV), Heliothis zea NPV, and Anticarsa gemmatalis NPV were compared with previously characterized granulins and polyhedrins. Similarities and differences were detected in the tryptic maps, while each protein produced a unique peptide map composite. Amino-terminal determination of eight polyhedrins and granulins resulted in three different end groups.     

Utilizable surface nutrients on Heliothis zea available for growth of Beauveria bassiana

Amino acids and glucosamine are present on the surface of Heliothis zea larvae. The amino acid compositions vary among instars and with time following completion of the gross molting process. Larvae collected from ears of corn have many surface amino acids. All amino acid combinations found on larval surfaces are sufficient for initiation of germination and growth by Beauveria bassiana. Amines and peptides are also present; these do not inhibit germination of B. bassiana or Aspergillus niger.     

A synchronous peroral technique for the bioassay of insect viruses

A relatively fast and simple peroral technique for the bioassay of insect viruses is described in which newly hatched larvae ingest a uniform volume of virus suspension. Three isolates of the Autographa californica nuclear polyhedrosis virus (NPV) and one isolate of the Heliothis zea NPV were used to test the procedure with Trichoplusia ni and H. zea larvae, respectively. Within-assay and between-assay variation was very low with coefficients of variation averaging 0.012 ± 0.006 and 0.20 ± 0.04 for time-mortality and dose-mortality tests, respectively. The synchronous uptake of virus removed the acquisition-time component of the LT₅₀ values while the constant volume improved the accuracy of LD₅₀ values. The procedure was shown to be suitable for a wide variety of lepidopterous species, including Spodoptera frugiperda, S. eridania, Estigmene acrea, Plutella xylostella, Choristoneura fumiferana, Ostrinia nubilalis, Plodia interpunctella, and Pieris rapae.